Absorbance of anthocyanin in beetroot experiment using spectrophotometric method

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Effect of alcohol on the diffusion of beetroot pigments measured using visible spectrophotometer. Please give proper reference to my IB student Gina on her work if you use this material

Effect of alcohol on the diffusion of beetroot pigments measured using visible spectrophotometer. Please give proper reference to my IB student Gina on her work if you use this material

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  • 1. IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067Candidate Name :Yoojin LeeCandidate Number :002213-067Date of Practical :March 14, 2010 Internal Assessment – Determining the relationship between ethanol concentration and rate of diffusion of Betanin pigment of beetroot, using the visible spectrophotometerResearch Question:How will changing ethanol concentration affect the rate of diffusion of beetroot pigment,Betanin1, from beetroot cubes placed in water, measured using visible spectrophotometer?Introduction:Visible spectrophotometer2 is a device that measures the absorbance of solutions. Somewavelengths of light pass through, but some wavelengths of light reflect back. For example,beetroot pigment, which is red in color, reflects wavelengths that code for red and absorbsother wavelengths that code for different colors. The detector records the reflection of light.Betanin is responsible for the red pigment in a beetroot. It is a glycoside composed of sugarand colored portion. It is water soluble, which lets diffusion possible in aqueous environment.Betanin is found in vacuoles in plant cells. When the plasma membrane of the plant cell isdenatured by ethanol, the Betanin pigments will flow out of the cell, down the concentrationgradient.The purpose of this experiment is to test the different absorbance at different concentration of1 George Pucher, Lawrence Curtis and Herbert Vickery, “The Red Pigment of the Root of the Beet (betaVulgaris),” The Journal of Biological Chemistry, http://www.jbc.org/content/123/1/61.full.pdf (accessed March14, 2010).2 “Ultraviolet–visible spectroscopy,” Wikipedia, the freeencyclopedia,http://en.wikipedia.org/wiki/Ultraviolet%E2%80%93visible_spectroscopy (accessed March 14,2010). 1
  • 2. IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067ethanol, hence find out the relationship between the rate of diffusion and the ethanolconcentration. The beetroot piece after washed with distilled water has its plasma membranesaround the cells to protect and to resist leaking of red pigments. However, when ethanol isadded to the solution surrounding the beetroot piece, ethanol molecules will destroy theplasma membranes and make red pigments to come out to the solution. In this experiment,the relationship between ethanol concentration and the rate of reaction, which is representedby absorbance of beetroot pigment, will be tested.The Beer-Lambert Law3 states A=ebc, in which A represents the absorbance, e representsmolar absorbtivity, b represents the path length of the cuvette, and c represents theconcentration of solution. In this experiment, the molar absorbtivity and cuvette are constant,because beetroot solution is the only solution to be tested and the same cuvette is used foreach trial. Thus, with two constant variables, the Beer-Lambert Law states that theabsorbance is directly proportional to the concentration. However, the linear relationshipbetween the absorbance and the concentration is deviated at high concentrations, so in thisexperiment, only solutions of low concentration are valid.3 “Beers Law,” Sheffield HallamUniversity,http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/beers1.htm (accessed March 14, 2010). 2
  • 3. IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067Hypothesis:Rate of diffusion is represented by the change in absorbance in an hour. Since ethanol has anability to destroy plasma membrane of beetroot cells, the increase in ethanol concentrationwill destroy the membranes more severely, which will result in excess diffusion of beetrootpigments from the cell. Thus, when the identically cut beetroot pieces are put into solutionswith different concentration, 0% ethanol solution will remain transparent, while 100%ethanol solution will have the darkest red color. When put into the visible spectrophotometer,ethanol solution will have absorbance value that is extremely close to 0, while 100% ethanolsolution will have the highest absorbance value. The relationship between ethanolconcentration and absorbance is directly proportional. Hence, as the ethanol concentrationgoes up, the rate of diffusion will increase accordingly.Figure 1 shows the predicted relationship between rate of diffusion and ethanol concentration 3
  • 4. IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067Variables: Variables Description Method of Measuring Independent Ethanol Concentration (%) 100% ethanol was diluted to 50% using distilled water. Then the diluted ethanol was further diluted to prepare 20%, 40%, 60%, 80%, and 100%. Distilled water was used for control (0% ethanol). Triplicate trials were performed on each concentration to obtain the mean. Dependant Rate of diffusion of Betanin Rate of diffusion is represented by the pigments from beetroot cells change of absorbance in an hour. Absorbance was measured using the visible spectrophotometer at λ max 480.5nm. Only one cuvette was used for each trial to reduce systematic errors. Controlled Size and type of beetroot Beetroot pieces of identical shape and size (0.5cm) was prepared using cork borer. Only the middle part of the beetroot was used. Same beetroot was used for all 3 trials. Size and type of cuvette The same cuvette for each trial was used, which was calibrated at the beginning of the trial. Volume of ethanol solution Ethanol and water to obtain was mixed carefully. Equal volume of the total solution, 2.5cm3, was prepared for all trials. Micropipette was used for accurate measurement. Time An hour was given for all trials for diffusion. Trials were simultaneously stopped by taking out beetroot pieces at the same time. Temperature Experiment was conducted in the lab at a constant room temperature, which is approximately 25℃. λ max λ max was fixed at 480.5nm, because the absorbance is measured relatively to the λ max. The maximum absorbance is determined by the λ max value. Table 1 shows the independent, dependent, and controlled variables and the methods of measuring 4
  • 5. IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067Apparatus: Materials:  Visible spectrophotometer  Beetroot  Micropipette (± 0.006cm3)  Ethanol (100%)  25cm3 Pipette (± 0.03cm3)  Distilled water  Microplate  Cuvette  Cork Borer (0.7cm diameter)  BeakerProcedure: 1. Using the cork borer, extract several strands of beetroot and cut the middle part, 0.5cm wide. 2. Put the beetroot pieces into distilled water to wash away pigments that are produced due to damages of plasma membrane. 3. Dilute 100% ethanol, using 25cm3 pipette. 4. Prepare different solutions of diluted ethanol in the microplate.Concentration, c/% Volume of diluted Volume of distilled Total Volume, ethanol, V1/cm3 water, V2/cm3 V/cm3 100 2.50 0.00 2.50 80.0 2.00 0.50 2.50 60.0 1.50 1.00 2.50 40.0 1.00 1.50 2.50 20.0 0.50 2.00 2.50 0.00 0.00 2.50 2.50 Table 2 shows the volume used to prepare solutions of different ethanol concentration 5. Place beetroot pieces into each solution using a tweezers and wait for an hour to let diffusion occur. 6. Calibrate cuvette and warm up the spectrophotometer at λ max 480.5nm 7. Using one cuvette for each trial, place the entire solution in the cuvette and measure the absorbance. 8. Repeat steps 6 and 7 to obtain the mean for the triplicate trials. 5
  • 6. IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067Data Collection and Processing:Quantitative Data Diluted Ethanol Absorbance at λ Max 480.5nm Concentration, Mean ± SD(b) c/% 1 2 3 Mean(a) 100 0.937 0.933 0.978 0.949 0.949 ± 0.020 80.0 0.902 0.854 0.905 0.887 0.887 ± 0.023 60.0 0.403 0.443 0.436 0.427 0.427 ± 0.017 40.0 0.070 0.068 0.082 0.073 0.073 ± 0.006 20.0 0.035 0.029 0.038 0.034 0.034 ± 0.004 0.00 0.043 0.029 0.045 0.039 0.039 ± 0.007 Table 3 shows mean absorbance of triplicate trials at λ max 480.5nm.(a) Mean: average of triplicate trials for each set.(b) SD: standard deviation for triplicate trials.Qualitative DataHigher ethanol solutions had redder and darker color than lower ethanol solutions. In fact, 0%ethanol concentration, which is distilled water, seemed transparent while 100% ethanolbecame darker as time went on. Since the Betanin pigment concentration was higher aroundthe beetroot, the solution had to be homogenized well before measuring the absorbance. 6
  • 7. IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067Data Processing Calculation of 100% ethanol concentration for the mean of triplicate trials. Mean ( ) = = Calculation of 100% ethanol concentration for the standard deviation of triplicate trials Standard deviation = = Calculation of 100% ethanol concentration for the rate of diffusion Rate of diffusion = =Subsequent calculations were performed on 80%, 60%, 40%, 20%, and 0%. Ethanol concentration, c/% Rate of diffusion, r/hr-1 100 0.949 ± 0.020 80.0 0.887 ± 0.023 60.0 0.427 ± 0.017 40.0 0.073 ± 0.006 20.0 0.034 ± 0.004 0.00 0.039 ± 0.007 Table 4 shows the relationship between the ethanol concentration and the rate of diffusion 7
  • 8. IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067Data Presentation: Rate of Diffusion,r/hr-1 against Different Concentration, c/% y = 0.0107x - 0.1316 of Diluted Ethanol at λ Max 480.5nm R² = 0.8739 1.2 1 (b) 0.8 Rate of Diffusion, r/hr-1 0.6 (a) 0.4 0.2 0 0 10 20 30 40 50 60 70 80 90 100 -0.2 Ethanol Concentration, c/% Graph 1 shows the rate of diffusion at λ max 480.5nm(a) Vertical error bar shows the standard deviation of the triplicate trials for the rate of diffusion(b) Horizontal error bar shows the uncertainty in ethanol concentration 8
  • 9. IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067Uncertainties:Diluting 100% ethanol % uncertainty for volume use Volume of Volume of 100% Total % distilled water ethanol using % uncertainty % uncertainty uncertainty, % using 25cm3 25cm3 pipette, in volume, % in volume, % pipette,(ΔV = ± 0.03)/cm3 (ΔV = ± 0.03)/cm3 (0.03/25) (0.03/25) 0.12 + 0.12 = 25 25 x100 = 0.12 x100 = 0.12 0.24 Table 5 shows the percent uncertainty for diluting ethanol. 9
  • 10. IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067 Uncertainty for volume use % uncertainty for volume use Ethanol Concentration Concentration Total %Concentration, with % with absolute uncertainty, %(c = ± 0.24)/% Volume of ethanol Volume of distilled water uncertainty, % uncertainty, %(a) % uncertainty % uncertainty using micropipette, using micropipette, in volume, % in volume, % (ΔV = ± 0.006)/cm3 (ΔV = ± 0.006)/cm3 (0.006/0.5) 0.24 + 6 + 0 100 2.50 x5x100 0.00 0.0 100±6.24 100±6.24 = 6.24 =6 (0.006/0.5) (0.006/0.5) 0.24 + 4.8 + 1.2 80.0 2.00 x4x100 0.500 x100 80.0±6.24 80.0±4.99 = 6.24 = 4.8 = 1.2 (0.006/0.5) (0.006/0.5) 0.24 + 3.6 + 2.4 60.0 1.50 x3x100 1.00 x2x100 60.0±6.24 60.0±3.74 =6.24 = 3.6 = 2.4 (0.006/0.5) (0.006/0.5) 0.24 + 2.4 + 3.6 40.0 1.00 x2x100 1.50 x3x100 40.0±6.24 40.0±2.50 = 6.24 = 2.4 = 3.6 (0.006/0.5) (0.006/0.5) 0.24 + 1.2 + 4.8 20.0 0.50 x100 2.00 x4x100 20.0±6.24 20.0±1.25 = 6.24 = 1.2 = 4.8 (0.006/0.5) 0.24 + 0 + 6 0.00 0.00 0.0 2.50 x5x100 0.00±6.24 0.00±0 = 6.24 =6 Table 6 shows percent uncertainty and absolute uncertainty for volume use for different ethanol concentrations (a) Absolute Concentration Calculation: 10
  • 11. IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067Conclusion:The data suggests that as the ethanol concentration increases, the rate of diffusion increasesand that my hypothesis is valid. The linear regression and the R2 value show that there is apositive correlation between the rate of diffusion and the ethanol concentration. However itcannot be proved that the correlation is always directly proportional. When observing the firstthree data on 0%, 20%, and 40% there was no obvious increases. Perhaps, this might be duethe fact that low ethanol concentration was not enough to destroy the plasma membraneeffectively. The graph slowly increased in the beginning and in the end, while the gradient inthe middle is extremely steep, which represents a huge change in absorbance in 60% ethanolrange. The results tell that when the ethanol concentration is high, the plasma membrane getsmore damaged, which leads to more out flux of Betanin pigment, which depends on theconcentration gradient. Thus, the results lead to a conclusion that the rate of diffusion isdirectly proportional when the ethanol concentration is higher than 40%.Evaluation:The experiment is justifiable because reliable triplicate trials were obtained. This is alsoreflected by the small standard deviation on graph 1. The uncertainty of the concentrationvaried with the concentration. Although it seems likely that there is a positive correlationbetween the rate of diffusion, according to graph 1, neither the 0% data nor the linearregression line pass through the origin of the data. This shows that both systematic andrandom errors were present.Since this experiment dealt with small pieces of beetroots and total of 2.5 cm3of solution pertrial, small systematic errors led to large uncertainties. For instance, an extra drop of ethanolcan change the percent concentration to a great extent. Major errors could have been reducedif bigger beet root samples with greater amount of ethanol solutions were used. 11
  • 12. IB Biology HL Name: Yoojin Lee Candidate Number: 002213-067Limitations and Improvements: Limitations ImprovementsThe beetroot pieces were not identical. Sincethe beetroot used for the experiment was verysmall, many strands could not be prepared. Prepare more strands of beetroot using theAlthough only middle pieces were used, it does cork borer and use only one middle partnot guarantee that every piece is the same. from each strand so that all of the pieces areEven though the beetroot pieces were different approximately the same.to a small extent, they could add highuncertainties, because only small amount ofsolutions were used.The beetroot pieces were not completelywashed after they were cut. There was no wayto be sure that the pigments on the surface of The beetroot pieces should be left longer. Itthe cut beetroot were washed away by will be faster if magnetic stirrer was used inobserving with naked eyes. Even if there were the process. However, a very small magneta method to check, it is impossible to check all has to be used to prevent damages onof the pieces simultaneously. Due to the time beetroot pieces.constraints, the beetroot pieces were washedfor 30 minutes.When mixing ethanol and water with Place the micro-pipette tip inside the watermicropipette, the solution formed bubbles that and transfer ethanol slowly to make themight have served as obstacles to measure the solutions homogenize smoothly.exact absorbance of the solution. Table 7 shows the limitations and the improvementsBibliography:Pucher, George, Lawrence Curtis, and Herbert Vickery. “The Red Pigment of the Root of the Beet (beta Vulgaris).” The Journal of Biological Chemistry.http://www.jbc.org/content/123/1/61.full.pdf (accessed March 14, 2010).“Beers Law.” Sheffield Hallam University.http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/beers1.htm (acces sed March 14, 2010).“Ultraviolet–visible spectroscopy.” Wikipedia, the free encyclopedia.http://en.wikipedia.org/wiki/Ultraviolet%E2%80%93visible_spectroscop y (accessed March 14, 2010). 12